Chlamydia species are an important cause of human disease for which no vaccine exists. Standard genetic approaches are not possible to employ with this obligate intracellular parasite, hampering our ability to decode its molecular pathogenesis. Recent transformative studies have revealed that despite its reduced genome size, on the order of 1000 genes, Chlamydia secretes well over 100 effector proteins into the host cell. These effectors function to selectively recruit organelles, acquire nutrients, manipulat host cell trafficking pathways, and to evade detection from the host immune system. Systematically characterizing the host proteins that are physically hijacked by pathogens that invade and replicate within the mammalian host cell is key to understanding their biology. Previous studies in simpler systems, including budding yeast and Escherichia coli, have demonstrated that such an endeavor is incredibly powerful with respect to uncovering novel biological insights. We propose two specific aims in which we will use state-of-the art high-throughput mass spectroscopy in conjunction with newly developed bioinformatic algorithms to (i) comprehensively identify host proteins that interact with secreted chlamydial effectors and to (ii) globally identify host post-translational modifications in response to chlamydial infections, including overall changes in the host proteome as well as changes in host protein phosphorylation, ubiquitylation, and glycosylation events. Aim 1. We will globally identify interactions between secreted C. trachomatis proteins and their host cell target proteins by affinity purification/mass spectrometry (AP/MS) to develop a comprehensive C. trachomatis-host protein-protein interaction (PPI) map. Aim 2. We will use an unbiased approach to globally identify changes in host post-translational modifications, including host protein phosphorylation, ubiquitylation and glycosylation, in response to C. trachomatis infection. Unraveling these complex events using these innovative approaches will yield important clues into the pathogenesis of Chlamydia respiratory infections, the role of Chlamydia infections in atherosclerotic heart disease, and the role of Chlamydia infections in lung cancer. The studies may provide new targets for diagnostics, therapeutics, and vaccines. In addition, these studies will provide novel insights into fundamental process in eukaryotic cell biology, with implications ranging from developmental biology to cancer biology.